In vitro testing is often used in the earlier stages of pre-clinical testing to eliminate unsafe compounds prior to advancement to the later animal stages. For example, interconnected cellular networks of cardiomyocytes may be formed on a substrate for the testing of potential new heart therapies. Primary cardiomyocytes harvested from an animal, or animal or human stem-cell-derived cardiomyocytes, form interconnected cellular networks when cultured on a cell culture substrate. The individual cardiomyocytes within a network are connected through gap junctions that allow ions to flow from one cell to another. This electrical connection allows an electrical action potential, which is first generated by a pacemaker cell, to propagate from one cell to the next.
Formation of an electrical action potential starts with a buildup of charge across a cell membrane. This buildup occurs spontaneously in cardiac cells, and more frequently in pacemaker cardiomyocytes than non-pacemaking cardiomyocytes. When the transmembrane charge reaches a threshold value, ions rush into the first cell (the depolarization phase). This triggers an action potential, which is a sharp influx of additional ions into the cytoplasm. The gap junctions distributed across the cell membrane allow ions to flow into neighboring cells, enabling the spread of the action potential.
Molecular processes within the cell tie the electrical action potential to the physical contraction of the cardiomyocytes. The propagation of the cardiac action potential across an in vitro cellular network, and the resulting contraction, resembles the propagation and contraction observed within the human heart and thus is often referred to as a “beat”. Many in vitro cardiomyocyte networks exhibit spontaneous beating, where each cardiac action potential propagation (and corresponding physical beat) is followed by a brief pause and then another cardiac action potential propagation and beat.
In vitro diagnostics allow researchers to analyze non-electrical properties of many types of cells, such as cell viability, density, and proliferation rates. However, electrically active cultures, such as cardiomyocytes, enable researchers to test additional properties related to electrical activity. For example, a cardiomyocyte culture may be assessed by the gap junction distribution, or degree of electrical connectivity between cells. This property may relate to the ability of a beat to be transmitted homogenously throughout a culture.
In another example, electrical measurements taken from a cardiomyocyte give researchers an indication of the cell health, quality, and level of maturity. For example, patch-clamp techniques provide measures of the action potential of an individual cell. A patch-clamp uses an electrode inserted into the cell membrane to measure transmembrane voltage. For a healthy cell, the cardiac action potential is initiated with a depolarization phase, where sodium rushes into the cell. The depolarization phase is followed by a plateau phase, dominated by the influx of calcium, where the cells remains depolarized, and ultimately a repolarization phase characterized by an outflux of potassium and a return to the starting transmembrane potential. Patch-clamp technologies can be used to detect abnormalities in the action potential within a single cell, which may point to functional problems. However, performing testing on individual cells is difficult and time-consuming. Furthermore, cardiomyocytes may behave differently when separated from their network, thus calling experimental results into question.
Other technologies, such as impedance measurement systems, can provide information about the physical beating of the cells, but do not reveal important functional information associated with the electrical action potential. Finally, optical imaging of the network electrophysiology can be performed using secondary voltage sensitive optical reporters. However, these protocols may be time consuming and cytotoxic, eliminating the ability to perform multiple experiments on the same culture.
Microelectrode arrays (MEAs) having a plurality of microelectrodes situated within each well enable researchers to measure signals from electrically active cells cultured on their surfaces. Herein, “microelectrode” and “electrode” will be used interchangeably. Cells are cultured across the array of electrodes within a well such that signals are detected from multiple electrically active cells, such as cardiomyocytes, simultaneously. These signals, called field potential signals, may change shape in response to the addition of a candidate compound to the cardiomyocyte culture. The changes may be used to evaluate the cardiac safety risk of a compound. Additionally, these measures may be used to develop and characterize new stem cell lines, to compare the electrophysiology of the cells to in vivo signals from native cardiomyocytes, and/or to evaluate in vitro models of disease.
In vitro electrophysiology culture systems having biosensors, MEAs, can provide important insights into networks of electrically active cells. MEA-based electrophysiology culture systems can be configured to concurrently monitor single-cell and network-level activity over extended periods of time and without affecting the cell culture under investigation. Since their instrumental role in the landmark discovery of spontaneous waves in a developing retina, the variety and scope of MEA-based electrophysiology applications has dramatically expanded. Recently, for example, MEA-based electrophysiology culture systems have been used to investigate the suppression of epileptic activity and in the study of novel plasticity mechanisms in cultured neural networks. Advances in cell culture preparations have similarly led to applications for MEA-based electrophysiology culture systems in the fields of drug screening, safety pharmacology, and biosensing.
Working with MEA-based systems requires physical access to the culture plate itself—for example, to setup, modify, or verify an ongoing experiment. One potential downside to physical access is the exposure of the culture plate and its contents to the ambient atmosphere. Typical ambient conditions are not ideal for cell growth and maintenance. For example, many cells prefer a Carbon dioxide (CO2)-rich environment that does not exist in the ambient atmosphere. Therefore, a scientist may inadvertently jeopardize the health of the cells any time they interact with the culture plate. A need exists for a system that allows physical and/or chemical interaction with cells in an MEA-based system while maintaining the accuracy of results (e.g., by preventing beat-period drift) and also minimizing harm to the cells themselves.
In order to accomplish these and other goals, Applicant has created various devices and systems for creating and maintaining a localized environment for a cell culture plate. A localized environment may describe any environment in proximity to the cell culture plate, and may include, for example, temperature, CO2 concentration, oxygen concentration, and so on.